Association of MMP-2-753C>T and MMP-9-1562C>T Polymorphisms with Chronic/Aggressive Periodontitis Risk: A Systematic Review and Meta-Analysis.

Background
Two functional polymorphisms in the matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9) genes may contribute to periodontitis pathogenesis. However, the results were inconsistent and inconclusive. Therefore, to clarify precise associations of MMP-2-753 C>T and MMP-9-1562C>T polymorphisms with chronic (CP) and aggressive (AgP) periodontitis, we performed a systematic review and meta-analysis.


Methods
A literature search was conducted using PubMed, Google Scholar, Embase, and Web of Science databases until 5 July 2017. The data were analyzed with CMA software, and risk estimates are expressed as odds ratios (ORs) and 95% confidence intervals (95% CIs).


Results
Nineteen case-control studies in ten publications with 2089 periodontitis cases and 2345 controls met the criteria. The pooled ORs indicated that MMP-2-753C>T and MMP-9-1562C>T polymorphisms were not significantly associated with risk of periodontitis in overall analysis. Stratified analyses by ethnicity and periodontitis type indicated that the MMP-9-1562C>T polymorphism showed a significant association with the risk of periodontitis among Caucasians and CP/AgP subgroup, whereas MMP-2-753C>T polymorphism was significantly associated with periodontitis risk only among Asians.


Conclusion
MMP-2-753C>T and MMP-9-1562C>T polymorphisms may not be associated with risk of periodontitis in overall population. However, MMP-2-753C>T and MMP-9-1562C>T polymorphisms might have influence on the susceptibility of periodontitis by ethnicity.


Introduction
Periodontitis is one of the most common causes of inflammatory bone loss in human (1)(2)(3). Perio-dontitis is a complex multifactorial disease that involves the interaction of environmental factors such as smoking and the patient's related factors such as sex, age and systemic diseases (1,4). Majority of the population has experienced some level of gingival inflammation worldwide, and 5%-8% of the population suffering from severe forms of periodontitis (5). Both chronic and aggressive forms of periodontitis are characterized by inflammation (6). Periodontitis as most human diseases have a genetic component, which influences inflammatory and immune responses in this disease (7). Results from the twin and family studies, indicate a role of genetic component in development periodontitis (8,9). Many studies have shown a correlation between periodontitis and systemic disease involving genes such as Matrix Metalloproteinases (MMP) as a shared mechanism of inflammation (8,10,11). Matrix metalloproteinases constitute a family of 25 zinc-dependent proteolytic enzymes, which are capable of degrading the extracellular matrix (ECM) (10)(11)(12). MMP-2 (Gelatinase-A) and -9 (Gelatinase-B) are two widely studied matrix metalloproteinases (13). MMP-2 gene (also known as 72-kDa type IV collagenase) is located on human chromosome 16q12.2 (14). MMP-2 gene encodes a protein that involved in the breakdown of ECM in normal physiological processes, such as embryonic development, reproduction and tissue remodeling (15). MMP-9 gene (also known as 92-kDa gelatinase or type V collagenase) is located on human chromosome 20q11.2-q13.1 (16). MMP-9 encodes a multidomain enzyme, a class of enzymes that belong to the zinc-metalloproteinases family involved in the degradation of the ECM (16,17). In the past decade, several epidemiologic studies have been investigated the association of MMP-9 -1562C>T (rs3918242) and MMP-2 -753C>T (rs2285053) polymorphisms with susceptibility to periodontitis (8,10,11). However, those studies results remain fairly inconsistent and inconclusive. A meta-analysis is a very powerful tool to obtain sufficient statistical power to detect the potential effect of these polymorphisms from individual studies with small size and the statistically low power.
Thus, we performed the current systematic and meta-analysis of all available case-control studies to provide more precise estimation of the association of MMP-2 -753C>T (rs2285053) and MMP-9 -1562C>T (rs3918242) polymorphisms with chronic/aggressive periodontitis susceptibility.

Inclusion and Exclusion Criteria
Studies were included based on the following criteria: 1) only full-text and published studies; 2) studies with case-control or cohort design; 3) a study evaluated the association of MMP-9 -1562C>T (rs3918242) and MMP-2 -753C>T (rs2285053) polymorphisms with periodontitis (CP and/or AgP) susceptibility risk; 4) available genotypes frequencies of MMP-9 -1562C>T (rs3918242) and MMP-2 -753C>T (rs2285053) polymorphisms were provided to estimate the odds ratios (ORs) with 95% confidence intervals (CIs). The exclusion criteria were as follows: 1) the study was not conducted on periodontitis; 2) abstracts, case reports, and review articles; 3) studies with only case group (no control group); 4) studies on the other polymorphisms of the MMP-2 and MMP-9 genes; 5) studies without detail genotype frequencies, which were unable to calculate ORs; and 6) duplicate publications of data from the same study.

Data Extraction
Two authors independently extracted the following data from each eligible study according to the inclusion criteria: the first author's name, the year of publication, ethnicity, country of origin, total number of cases and controls, the frequencies of genotypes, minor allele frequencies (MAFs), and Hardy-Weinberg equilibrium test in control subjects. We have calculated the allele frequencies from corresponding genotype distributions using an online website. Any discrepancy between these two authors was resolved by reaching a consensus through discussion or the involvement of a third author who made the final decision through discussions.

Statistical Analysis
Odds ratio (OR) and 95% confidence intervals (CIs) were calculated to evaluate the strength of the associations of MMP-9 -1562C>T and MMP-2 -753C>T polymorphisms with risk of periodontitis. The significance of the pooled OR was determined by the Z-test. Pooled ORs were performed for both MMP-9 -1562C>T and MMP-2 -753C>T polymorphisms under the allele model (T vs. C), the heterozygote model (TC vs. CC), the homozygote model (TT vs. CC), the dominant model (TT+TC vs. CC), and the recessive model (TT vs. TC+CC). Heterogeneity (betweenstudy inconsistency) was assessed by both the chi-square-based Q statistic (considered significant with P<0.10) and the I 2 statistic. In the current meta-analysis the I 2 values of 25, 50, and 75% meant a low, moderate, and high heterogeneity, respectively. When heterogeneity existed (P<0.10), a random-effects model weighted by the DerSimonian-Laird method was used to give a more conservative result; otherwise, a fixedeffects model weighted by the Mantel-Haenszel method would be applied. Sensitivity analyses were performed by omitting each particular study at a time. Hardy-Weinberg equilibrium (HWE) was evaluated for each study by Chi-square test in control groups, and P<0.05 was considered as a significant departure from HWE. Moreover, sensitivity analysis was also performed, excluding studies whose allele frequencies in controls exhibited significant deviation from the Hardy-Weinberg equilibrium (HWE), given that the deviation may denote bias. A meta-regression analysis was carried out to identify the major sources of between-studies variation in the results, using the log of the ORs from each study as ethnicity and types of periodontitis as the possible sources of heterogeneity. Moreover, the quality of selected studies was tested by the confirmation of HWE in control groups, and studies without the confirmation of HWE in controls were defined as low-quality studies, while studies with the confirmation of HWE in controls were defined as high-quality studies. Funnel plots and Egger's test were used to examine publication bias (P<0.05). If publication bias existed, the Duval and Tweedie non-parametric ''trim and fill'' method was used to adjust for it. The statistical analysis for the current meta-analysis study was performed using the Comprehensive Meta-Analysis (CMA) software (version 2.2; Biostat, USA). In the current meta-analysis, all P-values were considered two-sided, and P=0.05 was set as the threshold value for statistical significance.

Characteristics of the Included Studies
As shown in Fig. 1, the comprehensive search of literature under defined terms retrieved 197 articles. Of those 185 articles were excluded through duplicate screening and screening of titles and abstracts. Next, 45 studies were excluded because were reviews, case reports, irrelevance to the topic, not involving periodontitis research and lack-ing sufficient data. Finally, 19 case-control studies from ten publications (18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28) were identified in the meta-analysis. Basic characteristics of the selected articles are all listed in Table 1.   Table 1.

Quantitative Synthesis MMP-9 -1562C>T Polymorphism
The main results of MMP-9 -1562C>T polymorphism meta-analysis are shown in Table 2.

MMP-2 -753C>T Polymorphism
The main results of MMP-2 -753C>T polymorphism meta-analysis are shown in

Sensitivity Analysis
We have performed sensitivity analysis by omitting individual studies to assess the effect of each publication on the overall results. However, the significance of pooled ORs not influenced by omitting those studies, indicating that the results were stable. Additionally, we have performed sensitivity analysis by omitting six studies in which the genotype distributions of MMP-9 -1562C>T polymorphism in the healthy controls significantly deviated from the HWE. The results showed a significant association between MMP-9 -1562C>T polymorphism and periodontitis risk under recessive model (TT vs. TC+CC, OR=1.873, 95% CI=1.123-3.125, P=0.016), which suggests that the results of our metaanalysis are affected by HWE status.

Publication Bias
We have performed Begg's funnel plot and Egger's test to detect the publication bias of included studies. The shape of the funnel plot did not reveal any evidence of obvious asymmetry for MMP-2 -753C>T and MMP-9 -1562C>T polymorphisms under all genetic models. However, the results of Egger's test showed evidence of publication bias for MMP-9 -1562C>T under allele model (T vs. C: PBegg's=0.854, PEgger's=0.033). Then, we have used the Duval and Tweedie nonparametric ''trim and fill'' method to adjust the publication bias. However, meta-analysis with and without ''trim and fill'' did not draw different conclusion, indicating that our findings were statistically robust.

Discussion
In general, several studies revealed the relation between MMP-2 -753C>T and MMP-9 -1562C>T polymorphisms and susceptibility of periodontitis, however; the main findings from the different case-control studies did not reach the same conclusion. This inconsistency may result from the small sample size and the different experimental methods such as genotyping methods, ethnicity background, and subject's gender.
The present study showed that the MMP-2 -753C>T and MMP-9 -1562C>T polymorphisms were not associated with the susceptibility of periodontitis in overall analysis. However, there is still a need for further research and screening of etiological relations of the MMP-2 and 9 genes functional polymorphisms with the susceptibility of periodontitis. The limited statistical results may be reasonable due to the differences in the ethnic background, because different populations have different frequencies of alleles, and different genetic backgrounds may affect periodontitis risk. Therefore, we have performed subgroup analyses by ethnicity and disease type. The stratified analyses by ethnicity and periodontitis type revealed that the MMP-9 -1562C>T polymorphism showed a significant association with the risk of periodontitis among Caucasians and CP/AgP subgroup, whereas MMP-2 -753C>T polymorphism was significantly associated with periodontitis risk only among Asians. We found that the presence of T allele in MMP-2 -753C>T and MMP-9 -1562C>T polymorphisms is not significantly associated with an increased risk of periodontitis. Recently, two meta-analyses estimated the association between MMP-2 -753C>T and MMP-9 -1562C>T polymorphisms and periodontitis risk, which was basically in accordance with our results that MMP-2 -753C>T and MMP-9 -1562C>T polymorphisms may not contribute to the susceptibility of periodontitis in overall analysis (8,11). Moreover, at least four case-control studies (in two publications) (27,28) have not included in the meta-analysis (8). Additionally, Li et al. have included only two studies for MMP-2 -753C>T and MMP-9 -1562C>T polymorphisms (11). Thus, the ongoing uncertainty still exists and the conclusion by might be biased by not inclusion of new published studies (8,11). In our meta-analysis, we accurately assessed the association between these MMP-2 -753C>T and MMP-9 -1562C>T polymorphisms and the risk of chronic/aggressive periodontitis by taking into account the effects of new published studies. Moreover, a significant association had not found between MMP-2 -753C>T polymorphism and periodontitis according to disease type and ethnicity (8), while we have found that MMP-2 -753C>T polymorphism was significantly associated with periodontitis risk only among Asians. Therefore, both MMP-2 -753C>T and MMP-9 -1562C>T polymorphisms might have influence on the susceptibility of periodontitis by ethnicity background. Additionally, in the present metaanalysis we have provided the actually numbers of minor allele frequencies (MAFs) in the controls. The conclusion by our meta-analysis was more credible. Between-study heterogeneity is very common and expected in the genetic association studies of meta-analysis (29,30). Therefore, it is necessary to evaluate the magnitude of heterogeneity in a meta-analysis for determining the strengths of pooled results (31,32). We found relatively a high heterogeneity (>70%) for MMP-9 -1562C>T in overall analysis in all genetic models, but not for MMP-2 -753C>T polymorphism. We suggested that several factors including genetic backgrounds for cases and controls, diverse genotype distribution of MMP-2 -753C>T and MMP-9 -1562C>T polymorphisms in the included ethnicity groups, types of periodontitis (CP/AgP), dif-ferent genotyping methods, sample size of included studies, and uneven selection criteria for the cases and controls in different studies, responsible for such heterogeneity in our metaanalysis. Moreover, we have performed subgroup analysis by ethnicity, periodontitis type and HWE status to finding source of heterogeneity. The heterogeneity was reduced in the AgP group and also disappeared in Caucasians and Latinos populations, but not in the Asians and by HWE status.
In addition, we have found that that the heterogeneity was significantly reduced in the small sample size group in all genetic models, suggesting that the total sample size, ethnicity background, periodontitis type and HWE status were the sources of heterogeneity.
Although we performed a comprehensive metaanalysis, some limitations should be acknowledged. First, the number of published studies for MMP-2 -753C>T polymorphism was not sufficiently large for a comprehensive analysis, especially for stratified analyses by ethnicity. Second, the majority of the included studies was Asians or Caucasians, because of limited available data for MMP-2 -753C>T and MMP-9 -1562C>T polymorphisms from another ethnicity such as Africans, our results should be interpreted with caution. Larger studies are needed to clarify whether these two polymorphisms could truly affect the risk of periodontitis in other ethnicities. Third, our results were based on single-factor estimates without adjustment for other risk factors, therefore; our results may be affected by additional confounding factors, such as age, gender, smoking status, another chronic disease such as diabetes, caused serious confounding bias. If we had been able to acquire more detailed data, we would have achieved estimations that are more precise by adjusting for other potential covariates, but most of the included studies either did not report the data or aggregated them in different ways, making it impossible to include the data in the current meta-analysis. Therefore, studies with good design are needed in the future, and ORs adjusted for other confounding factors need reporting. Finally, it is also possible that language bias will exist, as in the present meta-analysis we have only included articles published in English. Finally, we have not addressed the effect of genegene and gene-environment interactions.

Conclusion
MMP-2 -753C>T and MMP-9 -1562C>T polymorphisms may not be associated with periodontitis risk in overall analysis. However, both MMP-2 -753C>T and MMP-9 -1562C>T polymorphisms might have influence on the susceptibility of periodontitis by ethnicity background.

Ethical considerations
Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.